Geranyl diphosphate synthase genes

ABSTRACT

The following recombinant protein (a) or (b) and a gene encoding the same:  
     (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:  1 ;  
     (b) a protein which consists of the amino acid sequence shown in SEQ ID NO:  1  having deletion, substitution or addition of at least one amino acid excluding the amino acid at position  82,  and which has geranyl diphosphate synthase activity.

TECHNICAL FIELD

[0001] The present invention relates to a geranyl diphosphate synthase,a gene encoding the synthase, a recombinant vector comprising the gene,and methods for preparing a geranyl diphosphate synthase and geranyldiphosphate, respectively.

BACKGROUND ART

[0002] Among those substances which have an important function inorganisms, there are a large number of substances biosynthesized withisoprene (2-methyl-1,3-butadiene) units. These compounds are also calledisoprenoids, terpenoids or terpenes. Depending on the number of carbonatoms they have, they are classified into hemiterpene (C5), monoterpene(C10), sesquiterpene (C15), diterpene (C20), sesterterpene (C25),triterpene (C30), tetraterpene (C40) and the like.

[0003] Actual biosynthesis of these substances starts with the synthesisof isopentenyl diphosphate (IPP), the active isoprene unit. Ultimately,the actual form of the isoprene unit which had been proposed as anputative precursor substance is IPP, the so-called active isoprene unit.

[0004] It is known that dimethylallyl diphosphate (DMAPP), an isomer ofIPP, is synthesized into an active isoprenoid such as geranyldiphosphate (GPP), neryl diphosphate, farnesyl diphosphate (FPP),geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP),hexaprenyl diphosphate (HexPP) or heptaprenyl diphosphate (HepPP),through condensation with IPP.

[0005] Through cis-condensation of FPP, GPP and the like in which theall-E type is considered to be the active type, a number of compoundssuch as natural rubber, dolichol, bactoprenol (undecaprenol) or variouspolyprenols found in plants are synthesized. It is considered that thesecompounds are synthesized by the consecutive condensation using theenergy of phosphate bonds between the pyrophosphoric acid and the carbonskeleton in their molecules. It is considered that pyrophosphoric acidis generated as a by-product of the condensation.

[0006] Active type isoprenoid synthases which condensate IPP intoallylic substrates of DMAPP, GPP, FPP, GGPP, GFPP, etc. in successionare called prenyl diphosphate synthases or prenyltransferases. Prenyldiphosphate synthases have different designations depending on thenumber of carbon atoms in their major reaction product. For example, theenzyme which catalyzes the production of farnesyl diphosphate with 15carbon atoms is called farnesyl diphosphate synthase (FPP synthase); andthe enzyme which catalyzes the production of geranylgeranyl diphosphatewith 20 carbon atoms is called geranylgeranyl diphosphate synthase (GGPPsynthase).

[0007] Various prenyl diphosphate synthase genes have already beenobtained from bacteria, archaea, fungi, plants and animals.Purification, activity determination as well as gene cloning and DNAsequencing have been reported on FPP synthases, GGPP synthases,hexaprenyl diphosphate synthases, heptaprenyl diphosphate synthases,octaprenyl diphosphate synthases, nonaprenyl diphosphate synthases(solanesyl diphosphate synthases), undecaprenyl diphosphate synthasesand the like.

[0008] These prenyl diphosphate synthases that are fundamental for thesynthesis of important and diversified compounds from both industrialand life-scientific viewpoints are generally unstable and low inspecific activity. Thus, industrial application of them could not beexpected. In recent several years, however, thermostable FPP synthasegenes and GGPP synthase genes have been isolated from thermophilicbacteria and archaea [A. Chen and D. Poulter (1993), J. Biol. Chem., 268(15), 11002-11007; T. Koyama et al., (1993), J. Biochem. (Tokyo), 113(3), 355-363; S.-i. Ohnuma et al., (1994), J. Biol. Chem., 269 (20),14792-14797]. Thus, conditions for utilizing prenyl diphosphatesynthases are now being prepared.

[0009] Enzymes which synthesize C₁ ₀₋₂ ₅ prenyl diphosphates arehomodimers. It is relatively easy to allow them to react in vitro, and anumber of reports have been made on their reaction. In those enzymes, anenzyme having activity to synthesize GPP (a C₁₀ prenyl diphosphate)specifically has not been isolated, though partial purification of ithas been reported (L. Heide and U. Berger, 1989, Arch, Biochem.Biophys., 273 (2) 331-8). Although it has been reported that a GPPsynthase was successfully purified from pig liver (J. K. Dorsey et al.,1966, J. Biol. Chem. 241 (22), 5353-5360), this enzyme catalyzes thesynthesis of FPP at the same time. Thus, based on the current definitionof prenyl diphosphate synthase, this enzyme should be called FPPsynthase.

[0010] GPP is the first intermediate for the synthesis of manymonoterpenes known and is the most important compound in thebiosynthesis pathway of monoterpenes.

[0011] Both geraniol and its isomer nerol, which are representativemonoterpenes, are aromatics in the major components of rose oil. Anotherrepresentative monoterpene camphor which is an extract from Cinnamomumcamphora is also used as a mothball.

[0012] However, GPP synthase gene has not been isolated yet.

[0013] Under circumstances, a technology is demanded which artificiallymodifies the amino acid sequence of a thermophile-derived, stable,homodimer type prenyl diphosphate synthase having a high specificactivity to thereby engineer a homodimer type, thermostable prenyldiphosphate synthase which specifically catalyzes the synthesis of GPP.

[0014] As thermophile-derived prenyl diphosphate synthases, Bacillusstearothermophilus FPP synthase and Sulfolobus acidocaldarius GGPPsynthase have been modified. Mutants of the S. acidocaldarius GGPPsynthase and genes thereof were selected using as an indicator anability to complement the glycerol metabolism ability of a HexPPsynthesis-dificient Saccharomyces serevisiae (budding yeast) [S.-i.Ohnuma et al., (1996), J. Biol. Chem., 271 (31), 18831-18837]. Mutantsof the S. stearothermophilus FPP synthase having GGPP synthesis activityand genes thereof were obtained using lycopene synthesis as an indicator[S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (17), 10087-10095].Further, 18 mutant enzymes which synthesize a number of prenyldiphosphates from GGPP to HexPP in various proportions, and genesencoding those enzymes were obtained by site-directed mutagenesis thenucleotides encoding the amino acid residue located 5 amino acidresidues upstream of the Asp-rich domain conserved region I (DDXX(XX)D)[S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (48), 30748-30754]. Ithas been found that the amino acid residue located 5 amino acid residuesupstream of the Asp-rich domain conserved region I (DDXX(XX)D) isinvolved in the regulation of chain lengths of reaction products.

[0015] However, no mutant enzyme having activity to synthesize GPPspecifically has been obtained yet.

DISCLOSURE OF THE INVENTION

[0016] It is an object of the invention to provide a geranyl diphosphatesynthase and a gene encoding the synthase.

[0017] As a result of extensive and intensive researches toward thesolution of the above problem, the present inventor has succeeded inisolating a geranyl diphosphate synthase and a gene encoding thesynthase by replacing a part of the amino acid sequence of a farnesyldiphosphate synthase. Thus, the present invention has been achieved.

[0018] The present invention relates to the following recombinantprotein (a) or (b):

[0019] (a) a protein consisting of the amino acid sequence shown in SEQID NO: 1;

[0020] (b) a protein which consists of the amino acid sequence shown inSEQ ID NO: 1 having deletion, substitution or addition of at least oneamino acid excluding the amino acid at position 82, and which hasgeranyl diphosphate synthase activity.

[0021] Further, the present invention relates to a gene coding for theabove-described recombinant protein (a) or (b).

[0022] Further, the present invention relates to a geranyl diphosphatesynthase gene comprising the nucleotide sequence shown in SEQ ID NO: 2.

[0023] Further, the present invention relates to a recombinant vectorcomprising any of the above-described genes.

[0024] Further, the present invention relates to a transformanttransformed with the above-described recombinant vector.

[0025] Further, the present invention relates to a method of preparing ageranyl diphosphate synthase comprising culturing the above-describedtransformant in a medium and recovering the geranyl diphosphate synthasefrom the resultant culture.

[0026] Further, the present invention relates to a method of preparinggeranyl diphosphate comprising culturing the above-describedtransformant in a medium and recovering geranyl diphosphate from theresultant culture.

[0027] Further, the present invention relates to a method of preparinggeranyl diphosphate comprising allowing a culture of the above-describedtransformant to act on isopentenyl diphosphate or an isomer thereof.

[0028] Hereinbelow, the present invention will be described in moredetail.

[0029] It is known that there are five conserved regions in the aminoacid sequence of a prenyl diphosphate synthase (if the synthase is aheterodimer, in the amino acid sequence of one of the sub-unit) [A. Chenet al. (1994) Protein Sci., 3 (4), 600-607]. In these five conservedregions (conserved regions I-V), there are two regions rich in asparticacid residues to which reaction products or reaction substrates arebelieved to be bound. These regions are called “aspartic acid richdomains” or “Asp-rich domains”. Of these, the Asp-rich domain located atthe amino terminal of prenyl diphosphate synthases (i.e. located in theabove-mentioned conserved region II) is designated Asp-rich domain I[sequence: DDXX(XX)D wherein the XX in parentheses may not exist], andthe Asp-rich domain located at the carboxyl terminal (i.e. located inthe above-mentioned conserved region V) is designated Asp-rich domain IIfor the purpose of discrimination.

[0030] Specific examples of prenyl diphosphate synthases containing suchaspartic acid rich domains as described above include farnesyldiphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyldiphosphate synthase, heptaprenyl diphosphate synthase, octaprenyldiphosphate synthase, nonaprenyl diphosphate synthase and undecaprenyldiphosphate synthase. As more specific examples, Bacillusstearothermophilus farnesyl diphosphate synthase, Escherichia colifarnesyl diphosphate synthase, Saccharomyces cerevisiae farnesyldiphosphate synthase, rat farnesyl diphosphate synthase, human farnesyldiphosphate synthase, Saccharomyces cerevisiae hexaprenyl diphosphatesynthase and the like may be enumerated. The amino acid sequences of theabove-mentioned conserved regions I-V in bacterial farnesyl diphosphatesynthases among those examples are shown in FIG. 4. In FIG. 4, “1.”represents the amino acid sequence of Bacillus stearothermophilusfarnesyl diphosphate synthase and “2.” represents the amino acidsequence of E. coli farnesyl diphosphate synthase. The portion enclosedwith a larger box shows Asp-rich domain I, and the portion marked with“⋆” shows the amino acid residue located 4 amino acid residues upstreamof this Asp-rich domain I.

[0031] The present invention is characterized by the creation of ageranyl diphosphate synthase by substituting the amino acid residuelocated 4 amino acid residues upstream of Asp-rich domain I with otheramino acid residue having a larger molecular weight than that residue,and by the preparation of geranyl diphosphate through the enzymereaction of the resultant geranyl diphosphate synthase. Morespecifically, a geranyl diphosphate synthase is created by substitutingthe amino acid residue marked with “⋆” in FIG. 4 (Ser) located 4 aminoacid residues upstream from the N-terminal amino acid (Asp) of thesequence DDXX(XX)D constituting Asp-rich domain I with other amino acidresidue having a larger molecular weight than Ser (any amino acidexcluding Gly and Ala; i.e. any amino acid selected from the groupconsisting of Val, Leu, Ile, Thr, Asp, Glu, Asn, Gln, Lys, Arg, Cys,Met, Phe, Tyr, Trp, His and Pro). The amino acid used for the abovesubstitution is not particularly limited as long as it is neither Glynor Ala. Preferably, Phe is used.

[0032] Specifically, the geranyl diphosphate synthase of the inventionis can be obtained by substituting the Ser residue at position 82 of theamino acid sequence shown in SEQ ID NO: 5 of a farnesyl diphosphatesynthase with, for example, Phe residue.

[0033] Such substitution can be achieved by partially modifying thenucleotide sequence of the gene encoding B. stearothermophilus FPPsynthase which is reported to be highly thermostable and high inspecific activity.

[0034] (1) Preparation of a Target Gene for Mutagenesis

[0035] A target gene into which a mutation is to be introduced is thegene encoding Bacillus stearothermophilus FPP synthase (hereinafterabbreviated to “BstFPS”). The full length nucleotide sequence of theBstFPS gene is known [T. Koyama et al., (1993) J. Biochem., 113,355-363; SEQ ID NO: 4] and is disclosed under Accession No. D13293 ingenetic information databases such as DDBJ.

[0036] Since B. stearothermophilus is also available from variousmicroorganism depositories such as ATCC (ATCC 10149), the DNA of theBstFPS gene can be obtained by conventional gene cloning methods [S.Sambrook et al. (eds.), (1989) Molecular Cloning, Cold Spring HarborLaboratory Press, New York].

[0037] Subsequently, the resultant DNA fragment is ligated to anappropriate plasmid vector (e.g. pTV118N from Takara Shuzo) to therebyprepare a plasmid DNA for mutagenesis. This plasmid DNA is designatedpFPS.

[0038] (2) Synthesis of an Oligonucleotide for Mutagenesis andIntroduction of a Mutation

[0039] An oligonucleotide for mutagenesis is designed so that (a) theSer codon corresponding to the amino acid residue at position 82 ofBstFPS is substituted with any codon (such as Phe codon) correspondingto any amino acid other than Gly, Ala and Ser; and (b) a restrictionsite for BspHI (5′TCATGA 3′) is newly introduced. For example, thefollowing nucleotide sequence may be given for the oligonucleotide.

[0040] 5′-CAT ACG TAC TTC TTG ATT CAT GAT GAT TTG-3′ (SEQ ID NO: 6)

[0041] This nucleotide sequence is designed so that the amino acidsequence encoded by the BstFPS gene is not altered by degeneracy ofcodons even after the introduction of the BspHI site. Because of theintroduction of this restriction site, it is possible to detect thoseplasmids into which a substitution mutation has been introduced byagarose gel electrophoresis of plasmid DNA after BspHI digestion.

[0042] The synthesis of the oligonucleotide may be performed withconventional chemical synthesis equipment. Preferably, the synthesizedoligonucleotide is phosphorylated and then denatured (for example, byheating it at 70° C. for 10 min).

[0043] Subsequently, using the oligonucleotide as a primer, a mutationis introduced into the plasmid prepared as described above. The methodof introduction of a mutation is not particularly limited. For example,a commercial kit based on the method of Kunkel [Proc. Natl. Acad. sci.,USA (1985) 82, 488] (Mutan-K kit from Takara Shuzo) may be used.Alternatively, polymerase chain reaction (PCR) may be used.

[0044] A single-stranded DNA is prepared as a template, and then theoligonucleotide described above was annealed with the template as acomplementary strand synthesis primer DNA to thereby obtain adouble-stranded DNA. The resultant DNA is incorporated into a plasmid,with which an E. coli strain is transformed.

[0045] The gene of the invention can be easily obtained, for example, byintroducing a mutation into the DNA encoding the native amino acidsequence of the synthase (SEQ ID NO: 4) by a conventional method such assite-directed mutagenesis or PCR.

[0046] For the resultant transformant clones, their nucleotide sequencesare determined. This determination may be performed by any conventionalmethod such as Maxam-Gilbert method or the dideoxy method. Usually, thedetermination is performed with an automated DNA sequencer based on thedideoxy method.

[0047] SEQ ID NO: 2 illustrates by way of example a nucleotide sequencefor the gene of the invention. SEQ ID NOS: 1 and 3 illustrate by way ofexample amino acid sequences for the geranyl diphosphate synthase of theinvention, which sequences may have a mutation such as deletion,substitution or addition of at least one amino acid (e.g. one or severalamino acids) excluding the amino acid at position 82 (e.g. Phe) as longas the protein consisting of the mutated sequence has geranyldiphosphate synthase activity. For example, the amino acid sequence ofSEQ ID NO: 1 or 3 in which the Met at position 1 is deleted is alsoincluded in the geranyl diphosphate synthase of the invention. Also, thegenes encoding these geranyl diphosphate synthases are also included inthe gene of the invention.

[0048] The “geranyl diphosphate synthase activity” used herein means acatalytic activity to synthesize GPP using IPP or an isomer thereof(e.g. DMAPP) as a substrate. The introduction of a mutation may beperformed by the same method as described above.

[0049] Once the nucleotide sequence of the geranyl diphosphate synthasegene of the invention has been established, the gene of the inventioncan be obtained by chemical synthesis, or by PCR using the gene as atemplate, or by hybridization using a DNA fragment having a nucleotidesequence of the gene as a probe.

[0050] (3) Construction of a Vector

[0051] A recombinant vector of the invention can be obtained by ligatingthe gene of the invention into an appropriate vector. The vector intowhich the gene of the invention is to be inserted is not particularlylimited as long as it is replicable in a host. A vector which may beused for the preparation of the recombinant vector of the invention canbe prepared “E. coli ” or the like by the alkali extraction method[Birnboim, H. C. & Doly, J. (1979) Nucleic Acid Res. 7: 1513) or avariation thereof. Alternatively, a commercial vector may be used as itis, or various vectors induced according to purposes may be used. Forexample, pBR322, pBR327, pKK233-2, pKK233-3 or pTrc99A having apMB1-derived replication origin may be enumerated. In addition, pUC18,pUC19, pUC118, pUC119, pTV118N, pTV119N, pBluescript, pHSG298 or pHSG396which is modified to give a greater number of copies, or a plasmidderived from pSC101, ColE1 factor, R1 plasmid or F factor may beenumerated. Further, a fusion protein expression vector such as pGEXvector or pMal vector which facilitates the purification of theexpressed product may be used.

[0052] It is also possible to perform gene transfer using a virus vector(e.g. λ phage or M13 phage) or a transposon instead of a plasmid. As aphage DNA, M13mp18, M13mp19, λ gt10, λ gt11 or the like may be used.

[0053] The incorporation of a DNA fragment encoding the geranyldiphosphate synthase into such a vector can be performed by conventionalmethods using an appropriate restriction enzyme and ligase. For example,a method may be employed in which a purified DNA is digested with anappropriate restriction enzyme and then inserted into the relevantrestriction site of an appropriate vector DNA for ligation.

[0054] The gene of the invention should be incorporated in the vector insuch a manner that the function of the gene can be manifested. For thispurpose, the vector of the invention may contain a replication originand expression regulating sequences appropriate for the host to be used.Further, the vector may also contain a transcription promoter,transcription terminator, ribosome binding site or the like. As thepromoter, Ptac, Plac or Ptrc may be used. As the terminator, rrnBterminator may be used. As the ribosome binding site, SD sequence(represented by 5′-AGGAGG-3′) may be used.

[0055] As a specific example of the thus prepared plasmid vector, pFPSmdescribed in Examples may be given.

[0056] (4) Preparation of a Transformant

[0057] A transformant of the invention can be obtained by introducingthe recombinant expression vector of the invention into a host so thatthe gene of interest can be expressed.

[0058] The host to be used is not particularly limited as long as it canexpress the gene of the invention. Specific examples of the host includeEscherichia or Bacillus bacteria such as E. coli , B. subtilis, B.brevis; Saccharomyces or Pichia yeasts such as S. cerevisiae , P.Pastris; filamentous fungi of the genus Aspergillus such as A. oryzae,A. niger; cultured cells of silkworm; animal cells such as COS cells orCHO cells; or plant cells.

[0059] When a bacterium such as E. coli is used as the host, preferably,the recombinant vector of the invention is capable of autonomousreplication in the host and, at the same time, is composed of atranscription promoter, a ribosome binding site, the DNA of theinvention and a transcription terminator. The vector may also contain agene to control the transcription promoter.

[0060] As the promoter sequence to start the transcription from DNA tomRNA, a native sequence (such as lac, trp, bla, lpp, PL, PR, T3 or T7)may be used. In addition to these promoters, mutants thereof (e.g.lacUV5) or sequences (e.g. tac, trc, etc.) in which a native promotersequence is artificially fused are known and may be used in the presentinvention.

[0061] With respect to a sequence which regulates the ability tosynthesize a protein from mRNA, it is already known that the distancebetween the ribosome binding site (GAGG and similar sequence) to theinitiation codon (ATG or GTG) is important. Further, it is well knownthat a terminator which commands the termination of transcription at the3′ end (e.g. rrnBT1T2) influences upon the protein synthesis efficiencyin a recombinant. Therefore, in the present invention, gene expressioncan be performed efficiently by using these sequences.

[0062] As a method for introducing a foreign gene into a bacterium, anymethod of DNA introduction into bacteria may be used. For example, amethod using calcium ions [Proc. Natl. Acad. Sci., USA, 69:2110-2114(1972)], electroporation or the like may be used.

[0063] When a yeast is used as the host, YEp13, YEp24, YCp50 or the likeis used as an expression vector. As a promoter used in this case, anypromoter may be used as long as it can direct the expression of the geneof interest in yeasts. For example, gall promoter, gal10 promoter, heatshock protein promoter, MF α 1 promoter or the like may be enumerated.

[0064] As a method for introducing a foreign gene into the yeast, anymethod of DNA introduction into yeasts may be used. For example,electroporation [Methods Enzymol., 194:182-187 (1990)], the spheroplastmethod [Proc. Natl. Acad. Sci., USA, 84:1929-1933 (1978)], the lithiumacetate method [J. Bacteriol., 153:163-168 (1983)] or the like may beenumerated.

[0065] When an animal cell is used as the host, pCDNAI/Amp, pcDNAL orthe like is used as an expression vector. In this case, the early genepromoter of human cytomegalovirus or the like may be used as a promoter.

[0066] As a method for introducing a foreign gene into the animal cell,electroporation, the calcium phosphate method, lipofection or the likemay be enumerated.

[0067] As a method for introducing a foreign gene into a plant cell, theinfection method using Agrobacterium is widely used. As a method fordirect introduction, the protoplast method, electroporation,bombardment, etc. may be enumerated.

[0068] The recombinant vector of the invention was incorporated into E.coli DH5α [designation: pFPSm(S82F)/DH5α] and deposited at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (1-3, Higashi 1-Chome, Tsukuba City, IbarakiPref., Japan) on Dec.12, 1997 as FERM BP-6551 under the Budapest Treaty.

[0069] (5) Production of the Geranyl Diphosphate Synthase

[0070] The geranyl diphosphate synthase of the invention can be obtainedby culturing the transformant described above in a medium and recoveringthe synthase from the resultant culture.

[0071] The cultivation of the transformant of the invention in a mediumis carried out by conventional methods used for culturing a host.

[0072] As a medium to culture the transformant obtained from amicroorganism host such as E. coli or yeast, either a natural orsynthetic medium may be used as long as it contains carbon sources,nitrogen sources and inorganic salts assimilable by the microorganismand enables effective cultivation of the transformant.

[0073] As carbon sources, carbohydrates such as glucose, fructose,sucrose, starch; organic acids such as acetic acid, propionic acid,citric acid; and alcohols such as glycerol, methanol, ethanol, propanolmay be used.

[0074] As nitrogen sources, ammonia; ammonium salts of inorganic ororganic acids such as ammonium chloride, ammonium sulfate, ammoniumacetate, ammonium phosphate; other nitrogen-containing compounds;Peptone; meat extract; corn steep liquor, etc. may be used.

[0075] As inorganic substances, potassium dihydrogen phosphate,dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate,magnesium chloride, sodium chloride, iron(II) sulfate, manganesesulfate, copper sulfate, calcium carbonate, calcium chloride, etc. maybe used.

[0076] When E. coli is used as a host, the cultivation is carried outusually under aerobic conditions (such as shaking culture or aerationagitation culture) at 37° C. for 16 to 24 hrs. During the cultivation,the pH is maintained at 6 to 8. The pH adjustment is carried out usingan inorganic or organic salt, an alkali solution, a buffer or the like.During the cultivation, an antibiotic such as ampicillin or tetracyclinemay be added to the medium if necessary.

[0077] When a microorganism transformed with an expression vector havingan inducible promoter is cultured, an inducer may be added to the mediumif necessary. For example, when a microorganism transformed with anexpression vector having lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added tothe medium. When a microorganism transformed with an expression vectorhaving trp promoter is cultured, indoleacrylic acid (IAA) or the likemay be added.

[0078] As a medium to culture a transformant obtained from an animalcell as a host, commonly used RPMI1640 medium or DMEM medium, or-one ofthese media supplemented with fetal bovine serum, etc. may be used.

[0079] Usually, the cultivation is carried out in the presence 5-10% CO₂at 37° C. for 2 to 20 days. During the cultivation, an antibiotic suchas kanamycin or penicillin may be added to the medium if necessary.

[0080] As a medium to culture a transformant obtained from a plant cellas a host, commonly used MS medium or this medium supplemented withkanamycin, various plant hormones, etc. is used. Usually, thecultivation is carried out at 20-30° C. for 3 to 14 days.

[0081] After the cultivation, the geranyl diphosphate synthase of theinvention is recovered by disrupting the microorganisms or cells if thesynthase is produced in the microorganisms or cells. If the geranyldiphosphate synthase of the invention is produced outside of themicroorganisms or cells, a culture supernatant is prepared by removingthe microorganisms or cells by centrifugation or the like. Then, thisculture (i.e. cell extract or culture supernatant) is subjected toconventional biochemical techniques used for isolating/purifyinng aprotein. These techniques include salting out, organic solventprecipitation, gel chromatography, affinity chromatography, hydrophobicinteraction chromatography and ion exchange chromatography. Thesetechniques may be used independently or in an appropriate combination tothereby isolate and purify the geranyl diphosphate synthase of theinvention from the culture.

[0082] It should be noted that the geranyl diphosphate synthase of theinvention can have geranyl diphosphate synthase activity even when it isnot purified from the culture. Therefore, the cell extract or culturefluid may be used as a crude enzyme solution without purification aslong as it has the synthase activity.

[0083] (6) Preparation of Prenyl Diphosphate

[0084] According to the present invention, it is possible to accumulateGPP in a culture by culturing the host transformed with the DNA of theinvention and to prepare GPP by recovering the accumulated GPP.

[0085] According to the present invention, it is also possible toprepare GPP by allowing the enzyme of the invention to act on IPP orDMAPP which is a substrate for the synthase. In this method, the enzymeof the invention is reacted with a reaction substrate in a solvent,particularly in an aqueous solution. Then, a prenyl diphosphate ofinterest is recovered from the reaction solution. As the enzyme, notonly a purified enzyme but also a crude enzyme which is semi-purified tovarious stages or an enzyme-containing material such as cultured cellsor a culture may also be used. Further, an immobilized enzyme which isobtained by immobilizing the above-mentioned enzyme, crude enzyme orenzyme-containing material by conventional methods may also be used.

[0086] As the substrate, IPP and/or DMAPP may be used. As the solventfor the reaction, water or an aqueous buffer such as Tris buffer orphosphate buffer may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

[0087]FIG. 1 is a graph showing the enzyme activity of mutant BstFPS andwild-type BsrFPS.

[0088]FIG. 2 is a photograph of thin-layer chromatogram.

[0089]FIG. 3 is a graph showing the reaction product specificity ofmutant BstFPS and wild-type BsrFPS.

[0090]FIG. 4 shows comparison of amino acid sequences of farnesyldiphosphate synthases.

BEST MODES FOR CARRYING OUT THE INVENTION

[0091] Hereinbelow, the present invention will be described morespecifically with reference to Examples. However, the technical scope ofthe invention is not limited to the following Examples.

[0092] Herein, amino acid residues are represented by the followingone-letter or three-letter abbreviations.

[0093] A; Ala; alanine

[0094] C; Cys; cysteine

[0095] D; Asp; aspartic acid

[0096] E; Glu; glutamic acid

[0097] F; Phe; phenylalanine

[0098] G; Gly; glycine

[0099] H; His; histidine

[0100] I; Ile; isoleucine

[0101] K; Lys; lysine

[0102] L; Leu; leucine

[0103] M; Met; methionine

[0104] N; Asn; asparagine

[0105] P; Pro; proline

[0106] Q; Gln; glutamine

[0107] R; Arg; arginine

[0108] S; Ser; serine

[0109] T; Thr; threonine

[0110] V; Val; valine

[0111] W; Trp; tryptophan

[0112] Y; Tyr; tyrosine

[0113] Herein, the substitution of an amino acid residue is expressedusing one-letter abbreviations in the following order: “the amino acidresidue before substitution”, “the position of the amino acid residue”and “the amino acid residue after substitution”.

[0114] For example, when Ser at position 82 is substituted with Phe,this substitution is expressed as “S82F”.

EXAMPLE 1 Preparation of a Plasmid Comprising FPP Synthase Gene

[0115]Bacillus stearothermophilus-derived FPP synthase (BstFPS) gene wassub-cloned into the NcoI-HindIII site of a plasmid vector pTV118N(commercially available from Takara Shuzo). This plasmid DNA isdesignated pFPS. The full-length nucleotide sequence of BstFPS gene isdisclosed by T. Koyama et al., (1993) J. Biochem., 113, 355-363 or ingenetic information databases such as DDBJ under Accession No. D13293.

EXAMPLE 2 Synthesis of an Oligonucleotide for Mutagenesis

[0116] The following oligonucleotide was synthetized in order tointroduce a mutation into the gene obtained in Example 1.

[0117] 5′-CAT ACG TAC TTC TTG ATT CAT GAT GAT TTG-3′ (SEQ ID NO: 6)

[0118] The above oligonucleotide is designed so that (a) the Ser codoncorresponding to the amino acid residue at position 82 of BstFPS issubstituted with Phe codon; and (b) a restriction site for BspHI(5′TCATGA 3′) is newly introduced. The introduction of this BspHI sitedoes not cause any alteration due to degeneracy of codons in the aminoacid sequence encoded by BstFPS gene. Because of the introduction ofthis restriction site, it is possible to detect those plasmids intowhich a substitution mutation has been introduced by agarose gelelectrophoresis of plasmid DNA after BspHI digestion.

[0119] The synthesized oligonucleotide was phosphorylated in thefollowing reaction solution at 37° C. for 30 min and then inactivated at70° C. for 10 min. 10 pmol/μl oligonucleotide 2 μl 10 x kination buffer1 μl 1000 mM Tris-Cl (pH 8.0)  100 mM MgCl₂  70 mM DTT 10 mM ATP 1 μlH₂O 5 μl T4 polynucleotide kinase 1 μl

EXAMPLE 3 Introduction of Substitution Mutation into the CodonCorresponding to the Amino Acid Residue at Position 82 of BstFPS Gene

[0120] Using the oligonucleotide synthesized in Example 2 as a primer, asubstitution mutation was introduced into the plasmid prepared inExample 1 according to the method of Kunkel. In the practice of thismethod, Mutan-K kit commercially available from Takara Shuzo was used.Experimental procedures were according to the protocol attached to thekit.

[0121] Briefly, a single-stranded DNA in which thymine in plasmid pFPSDNA had been replaced with deoxyuracil was prepared using E. coli CJ-236as a host cell.

[0122] With this single-stranded DNA as a template, the primer DNA forcomplementary strand synthesis (i.e. the above oligonucleotide) wasannealed in the following solution. Single-stranded DNA 0.6 pmolAnnealing buffer 1 μl  200 mM Tris-Cl (pH 8.0) 100 mM MgCl₂ 500 mM NaCl 10 mM DTT Primer DNA (from Example 2) 1 μl  H₂O to give a final volumeof 10 μl

[0123] Subsequently, 25 μl of extension buffer, 60 units of E. coli DNAligase and 1 unit of T4 DNA polymerase were added to the solution and tosynthesize a complementary strand at 25° C. for 2 hrs. The extensionbuffer was composed of 50 mM Tris-Cl (pH 8.0), 60 mM ammonium acetate, 5mM MgCl₂, 5 mM DTT, 1 mM NAD and 0.5 mM dNTP.

[0124] Then, the reaction was terminated by adding thereto 3 μ1 of 0.2 MEDTA (pH 8.0) and treating the resultant solution at 65° C. for 5 min.

EXAMPLE 4 Creation of a Transformant Whose Gene Has SubstitutionMutation in the Codon Corresponding to the Amino Acid Residue atPosition 82 of BstFPS Gene

[0125]E. coli DH5α a was transformed with the DNA solution prepared inExample 3 by the calcium chloride method as described below. Briefly,the DNA solution was added to a suspension of DH5α competent cellstreated with 50 mM CaCl₂. Then, the suspension was put on ice for 30min.

[0126] The resultant transformants were plated on an agar platecontaining ampicillin (a transformant selection marker), and cultured at37° C. overnight. Plasmid DNA was prepared from a transformant havingampicillin resistance as a phenotype. After digestion with BspHI, theplasmid DNA was subjected to agarose gel electrophoresis to therebyselect substitution mutant pFPS plasmid which has a BspHI site withinthe BstFPS coding region from the resultant transformants.

[0127] Subsequently, the nucleotide sequence around the codoncorresponding to the amino acid residue at position 82 of BstFPS gene inthe selected substitution mutant pFPS plasmid was determined by thedideoxy method. As a result, a pFPS plasmid comprising a substitutionmutant BstFPS gene (SEQ ID NO: 2) in which the Ser codon at position 82(TCT) had been replaced with Phe codon (TTC) was obtained. This mutantis designated S82F, and the plasmid pFPSm.

EXAMPLE 5 Determination of the Activity of Mutant BstFPS

[0128] Crude enzyme solutions were prepared as described below from twotransformants comprising the mutant BstFPS gene obtained in Example 4and wild-type BstFPS gene, respectively, and a transformant comprisingvector pTV118N alone.

[0129] Cells of each transformant cultured overnight in 2x LB mediumwere harvested by centrifugation and suspended in a cell disruptionbuffer [50 mM Tris-Cl (pH 8.0), 10 mM β-mercaptoethanol, 1 mM EDTA).This suspension was sonicated and then centrifuged at 4° C. at 10,000r.p.m. for 10 min. The resultant supernatant was thermally treated at55° C. for 30 min to inactivate the prenyl diphosphate synthases derivedfrom E. coli. The thus treated supernatant was centrifuged under thesame conditions as described above to obtain a supernatant as a crudeenzyme extract. This enzyme extract was reacted at 55° C. for 15 min inthe following reaction solution. [1⁻¹⁴ C]-IPP (1 Ci/mol) 25 nmol Allylicdiphosphate (DMAPP or GPP or FPP) 25 nmol Tris-Cl (pH 8.5) 50 mM MgCl₂ 5mM NH₄Cl 50 mM β-mercaptoethanol 50 mM Enzyme solution 50 μl H₂O to givea total volume of 1 ml

[0130] After the reaction, 3 ml of water-saturated butanol was added tothe reaction solution to extract the reaction products into the butanollayer. To 1 ml of the resultant butanol layer, 3 ml of a liquidscintillator was added. Then, the mixture was subjected to thedetermination of radioactivity using a liquid scintillation counter.

[0131] The results are shown in FIG. 1. FIG. 1 is a graph showing theenzyme activity of S82F mutant BstFPS and wild-type BsrFPS. Sample Nos.1, 4 and 7 represent an enzyme prepared from a host comprising vectorpTV118N alone. Sample Nos. 2, 5 and 8 represents an enzyme prepared froma host comprising a gene encoding S82F mutant BstFPS. Sample Nos. 3, 6and 9 represent an enzyme prepared from a host comprising a geneencoding wild-type BstFPS. Further, sample Nos. 1, 2 and 3 represent theresults when DMAPP was used as an allylic substrate. Sample Nos. 4, 5and 6 represent the results when FPP was used as an allylic substrate.Sample Nos. 7, 8 and 9 represent the results when FPP was used as anallylic substrate.

[0132] From FIG. 1, it is understood that the wild-type enzyme can useDMAPP and GPP as an allylic substrate but cannot use FPP. On the otherhand, it is shown that the ability to use GPP as an allylic substrate isextremely lowered in S82F mutant enzyme.

[0133] Subsequently, a reaction solution was prepared separately in thesame manner as described above. Immediately after the reaction, 1 ml ofa potato acid phosphatase solution [2 mg/ml potato acid phosphatase, 0.5M sodium acetate (pH 4.7)] was added to the reaction solution, which wasdephosphorylated at 37 ° C. and then extracted with 3 ml of pentane. Theextract was analyzed by thin-layer chromatography [reversed phase TLCplate:LKC18 (Whatman); developer:acetone/water=9/1]. The developed,dephosphorylated reaction products were applied to Bioimage AnalyzerBAS2000 (Fuji Photo Film) to determine the positions and relativequantities of radioactivity.

[0134] The results are shown in FIGS. 2 and 3. FIG. 2 shows the TLCdevelopment patterns of the dephosphorylated, mutant PstFPS reactionproducts when individual allylic substrates were used. For comparison,patterns obtained from samples prepared from hosts comprising wild-typeBstFPS gene and a vector alone, respectively, are also shown. In thisfigure, “s.f.” represents the solvent front; “ori” represents thedevelopment origin; “GOH” represents the position of geraniol standardsample developed; and “FOH” represents the position of farnesol standardsample developed. “Wild type” shows the results when wild-type BstFPSwas used; “S82F” shows the results when mutant S82F BstFPS was used; and“vector” shows the results when an enzyme prepared from a hostcomprising a vector alone was used. “n.d.” means that activity was notdetected. FIG. 3 is a graph showing the reaction product specificity ofwild-type BstFPS and mutant BstFPS. This graph shows GGPP, FPP and GPPgeneration ratios when IPP and DMAPP were used as a substrate.

[0135] From the results shown in FIGS. 2 and 3, it is understood thatwhile wild-type BstFPS catalyzes a reaction to synthesize FPPspecifically, S82F mutant BstFPS has been changed to catalyze a reactionto synthesize GPP specifically. This means that S82F mutant BstFPS hasbeen changed to an enzyme that can be called a geranyl diphosphatesynthase.

[0136] Industrial Applicability

[0137] According to the present invention, a geranyl diphosphatesynthase, a gene encoding the synthase, a recombinant vector comprisingthe gene, and methods for preparing a geranyl diphosphate synthase andgeranyl diphosphate, respectively, are provided.

[0138] The gene of the invention is useful since it is applicable tometabolic engineering and enzyme engineering aiming at the synthesis ofmonoterpenes.

[0139] Free Text to the Sequence Listing

[0140] SEQ ID NO: 1: Xaa represents Val, Leu, Ile, Thr, Asp, Glu, Asn,Gln, Lys, Arg, Cys, Met, Phe, Tyr, Trp, His or Pro.

[0141] SEQ ID NO: 6: Oligonucleotide which is designed based on theamino acid sequence of FPP synthase and has a BspHI site.

1. The following recombinant protein (a) or (b): (a) a proteinconsisting of the amino acid sequence shown in SEQ ID NO: 1; (b) aprotein which consists of the amino acid sequence shown in SEQ ID NO: 1having deletion, substitution or addition of at least one amino acidexcluding the amino acid at position 82, and which has geranyldiphosphate synthase activity.
 2. A gene coding for the followingrecombinant protein (a) or (b): (a) a protein consisting of the aminoacid sequence shown in SEQ ID NO: 1; (b) a protein which consists of theamino acid sequence shown in SEQ ID NO: 1 having deletion, substitutionor addition of at least one amino acid excluding the amino acid atposition 82, and which has geranyl diphosphate synthase activity.
 3. Ageranyl diphosphate synthase gene comprising the nucleotide sequenceshown in SEQ ID NO:
 2. 4. A recombinant vector comprising the gene ofclaim 2 or 3 .
 5. A transformant transformed with the recombinant vectorof claim 4 .
 6. A method of preparing a geranyl diphosphate synthasecomprising culturing the transformant of claim 5 in a medium andrecovering the geranyl diphosphate synthase from the resultant culture.7. A method of preparing geranyl diphosphate comprising culturing thetransformant of claim 5 in a medium and recovering geranyl diphosphatefrom the resultant culture.
 8. A method of preparing geranyl diphosphatecomprising allowing a culture of the transformant of claim 5 to act onisopentenyl diphosphate or an isomer thereof.